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The generation of human hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) represents a major goal in regenerative medicine and is believed would follow principles of early development. HSCs arise from a type of endothelial cell called a “hemogenic endothelium” (HE), and human HSCs are experimentally detected by transplantation into SCID or other immune-deficient mouse recipients, termed SCID-Repopulating Cells (SRC). Recently, SRCs were detected by forced expression of seven transcription factors (TF) (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and SPI1) in hPSC-derived HE, suggesting these factors are deficient in hPSC differentiation to HEs required to generate HSCs. Here we derived PECAM-1-, Flk-1-, and VE-cadherin-positive endothelial cells that also lack CD45 expression (PFVCD45−) which are solely responsible for hematopoietic output from iPSC lines reprogrammed from AML patients. Using HEs derived from AML patient iPSCs devoid of somatic leukemic aberrations, we sought to generate putative SRCs by the forced expression of 7TFs to model autologous HSC transplantation. The expression of 7TFs in hPSC-derived HE cells from an enhanced hematopoietic progenitor capacity was present in vitro, but failed to acquire SRC activity in vivo. Our findings emphasize the benefits of forced TF expression, along with the continued challenges in developing HSCs for autologous-based therapies from hPSC sources.

The expanding field of hematopoietic cell transplantation (HCT) for non-malignant diseases, including those amenable to gene therapy or gene editing, faces challenges due to limited donor availability and the toxicity associated with cell collection methods. Umbilical cord blood (CB) represents a readily accessible source of hematopoietic stem and progenitor cells (HSPCs); however, the cell dose obtainable from a single cord blood unit is frequently insufficient. This limitation can be addressed by enhancing the potency of HSPCs, specifically their capacity to reconstitute hematopoiesis. In our study, we investigated the combined effects of treprostinil, a prostaglandin analog, and cinacalcet, a calcium-sensing receptor modulator, on the reconstitution of hematopoiesis. A Lineage Cell Depletion Kit was employed to isolate lineage-negative (lin ) HSPCs from mouse bone marrow. A Human CB CD34 Positive Selection Kit was utilized to isolate CD34 cells from the CB of healthy donors. , the effects of treprostinil, cinacalcet, and their combination on the migration, adhesion, and differentiation of HSPCs were assessed. , homing and engraftment were examined. Eight-week-old female and male C57BL/6J, BALB/c, or female NSG mice served as recipient models. When administered concomitantly, treprostinil and cinacalcet exhibited mutual antagonism: the survival of recipient animals was lower when both drugs were administered together compared to either agent alone. Conversely, a sequential regimen involving priming with treprostinil/forskolin followed by cinacalcet treatment enhanced survival, irrespective of whether hematopoiesis was reconstituted by human or murine HSPCs. assays demonstrated enhanced migration and adhesion in response to the presence of treprostinil and cinacalcet, suggesting potential synergistic effects. Colony formation confirmed synergism. Augmenting the bone marrow reconstitution potential of HSPCs with treprostinil and cinacalcet shows promise for rescuing patients undergoing HCT. This approach is particularly beneficial for those patients at high risk of transplant failure due to limited numbers of available HSPCs. Furthermore, enhancing the potency of HSPCs has the potential to alleviate the burden and risks associated with HSPC donation, as it would reduce the number of cells needed for collection.

Hematopoietic stem and progenitor cells (HSPCs) mobilization is the movement of HSPCs from the bone marrow to the peripheral blood or tissue induced by stress. HSPC mobilization is a well-known response to protect the host during infection through urgent differentiation of HSPCs to immune cells. Dengue virus (DENV) infection is known to cause stress in infected humans and the mobilizing capacity of HSPCs during DENV infection in affected patients has not been fully investigated. Here, we investigated whether DENV infection can induce HSPC mobilization and if the mobilized HSPCs are permissive to DENV infection. White blood cells (WBCs) were collected from dengue patients (DENV+) and healthy donors and analyzed by flow cytometry and plaque assay. Elevated HSPCs levels were found in the WBCs of the DENV+ group when compared to the healthy group. Mobilization of HSPCs and homing markers (skin and gut) expression decreased as the patients proceeded from dengue without symptoms (DWoWS) to severe dengue (SD). Mobilizing HSPCs were not only permissive to DENV infection, but infectious DENV could be recovered after coculture. Our results highlight the need for further investigation into HSPC mobilization or alterations of hematopoiesis during viral infections such as DENV in order to develop appropriate countermeasures.

During vertebrate embryogenesis, fetal hematopoietic stem and progenitor cells (HSPCs) exhibit expansion and differentiation properties in a supportive hematopoietic niche. To profile the developmental landscape of fetal HSPCs and their local niche, here, using single-cell RNA-sequencing, we deciphered a dynamic atlas covering 28,777 cells and 9 major cell types (23 clusters) of zebrafish caudal hematopoietic tissue (CHT). We characterized four heterogeneous HSPCs with distinct lineage priming and metabolic gene signatures. Furthermore, we investigated the regulatory mechanism of CHT niche components for HSPC development, with a focus on the transcription factors and ligand–receptor networks involved in HSPC expansion. Importantly, we identified an endothelial cell-specific G protein–coupled receptor 182, followed by in vivo and in vitro functional validation of its evolutionally conserved role in supporting HSPC expansion in zebrafish and mice. Finally, comparison between zebrafish CHT and human fetal liver highlighted the conservation and divergence across evolution. These findings enhance our understanding of the regulatory mechanism underlying hematopoietic niche for HSPC expansion in vivo and provide insights into improving protocols for HSPC expansion in vitro.

Umbilical cord blood mononuclear fraction is a valuable source of hematopoietic stem and progenitor cells (CB HSPCs). The rarity of this population is a serious limitation of its application in cell therapy. Ex vivo expansion enables to significantly amplify the number of hematopoietic precursors of different commitment. Here, we expand CB MNCs in co-culture with human adipose tissue-derived stromal cells (ASCs) to enrich HSPCs and describe phenotypic features of newly formed hematopoietic populations. The CD34+-HSPCs demonstrated 6-fold enrichment with 9000 CFUs per 50 × 103 HSPCs on average. A part of the floating HSPCs were bearing lineage markers, while others were primitive precursors (CD133−/CD34+). Among ASC-associated HSPCs, two subsets of cord blood-borne cells were revealed: СD90+/СD45− and СD90+/СD45+. The proportion of CD3+/CD8+ and NK-T as well as CD25+ and HLA-DR+ Т cells among СD90+/СD45− cells was significantly higher compared to MNCs and floating HSPCs. More than 80% of CD45+/СD90+ HSPCs were identified as late primitive precursors (CD133−/CD34+). Thus, CB MNC expansion in the presence of ASCs provides the generation of both lineage committed lymphoid progenitors and CD34+/CD133− primitive HSPCs. Substantially enriched with primitive precursors, ASC-associated HSPCs could be considered as a perspective tool for a long-term restoration of hematopoiesis in various hematologic disorders.

CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as β-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because β-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.

Hematopoietic stem and progenitor cells (HSPCs), residing at the apex of the hematopoietic hierarchy, are critical for the generation of all blood and immune cell lineages. This unique capacity makes HSPCs indispensable for advancing cell and gene therapies aimed at correcting defects across hematopoietic lineages. However, current gene therapy development is constrained by the requirement for fresh primary HSPCs, hindering the breadth of preclinical validation. While several hematopoietic lineages have been immortalized to facilitate research, the stable immortalization of human HSPCs remains unreported. Here, we demonstrate that combinatorial overexpression of HLF, a key regulator of stem cell maintenance, and hTERT, a telomere maintenance factor in primitive human HSCs, supported by BaEV-mediated transduction and optimized culture conditions, yields partial immortalization of HSPCs. These genetically modified cells sustained for up to 70 days and exhibited limited differentiation towards erythroid, megakaryocytic, and macrophage lineages. Our model establishes a protocol for generating primary cell like context for testing gene therapy strategies, enabling functional assessment in both undifferentiated HSPCs and their lineage-committed progeny.

Chronic inflammatory diseases are associated with hematopoietic lineage bias, including neutrophilia and anemia. We have recently identified that the canonical inflammasome mediates the cleavage of the master erythroid transcription factor GATA1 in hematopoietic stem and progenitor cells (HSPCs). We report here that genetic inhibition of Nlrp1 resulted in reduced number of neutrophils and increased erythrocyte counts in zebrafish larvae. We also found that the NLRP1 inflammasome in human cells was inhibited by LRRFIP1 and FLII, independently of DPP9, and both inhibitors regulated hematopoiesis. Mechanistically, erythroid differentiation resulted in ribosomal stress‐induced activation of the ZAKα/P38 kinase axis which, in turn, phosphorylated and promoted the assembly of NLRP1 in both zebrafish and human. Finally, inhibition of Zaka with the FDA/EMA‐approved drug Nilotinib alleviated neutrophilia in a zebrafish model of neutrophilic inflammation and promoted erythroid differentiation and GATA1 accumulation in K562 cells. In conclusion, our results reveal that the NLRP1 inflammasome regulates hematopoiesis and pave the way to develop novel therapeutic strategies for the treatment of hematopoietic alterations associated with chronic inflammatory and rare diseases. Synopsis Deregulation of inflammasome activity contributes to hematopoietic alterations associated to chronic inflammatory diseases, including neutrophilia and anemia. Zebrafish models and human blood cells were used to identify the critical role of the NLRP1 inflammasome on the regulation of hematopoiesis and possible therapeutic targets for clinical intervention. Erythroid differentiation resulted in ribosomal stress‐induced activation of the ZAKα/P38 kinase axis. ZAKα/P38 kinase axis phosphorylated and promoted the assembly of NLRP1 in HSPCs which, in turn, regulates their erythroid‐myeloid lineage decision. NLRP1 inflammasome in HSPCs was inhibited by LRRFIP1 and FLII, independently of DPP9. The FDA/EMA‐approved drug Nilotinib inhibited NLRP1 inflammasome activation alleviating neutrophilia and promoting erythroid differentiation. Graphical Abstract Deregulation of inflammasome activity contributes to hematopoietic alterations associated to chronic inflammatory diseases, including neutrophilia and anemia. Zebrafish models and human blood cells were used to identify the critical role of the NLRP1 inflammasome on the regulation of hematopoiesis and possible therapeutic targets for clinical intervention.

Background After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences. Results We show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs). We find that the homology-directed repair (HDR) with single-stranded oligodeoxynucleotides (ssODNs) carrying short homology reduces the deletion damage by almost half, while adeno-associated virus (AAV) donors with long homology reduce large deletions by approximately 80%. In the absence of HDR, the insertion of a short double-stranded ODN by NHEJ reduces deletion indexes by about 60%. Conclusions Timely bridging of broken ends by HDR and NHEJ vastly decreases the unintended consequences of dsDNA cleavage. These strategies can be harnessed in gene editing applications to attenuate unintended outcomes.

Abstract Objectives The intercorrelations between periodontitis and inflammatory bowel disease have been recognized for years. Accumulating evidence has shown that patients with ulcerative colitis (UC) have a higher prevalence and severity of periodontitis. However, the underlying mechanisms by which UC aggravates periodontal destruction are still unclear. Methods Multiple murine models, including DSS-induced colitis (DIC)/ligature-induced periodontitis (LIP), DIC/LIP rescued by berberine, and LIP after DIC remission models were established to investigate the mechanisms by which UC exacerbates periodontal inflammation. Results DIC mice exhibited a disrupted intestinal barrier with dysbiotic gut microbiota, corroborating the elevated serum levels of LPS and IL-1. Compared to DIC-free/LIP mice, DIC/LIP mice showed aggravated alveolar bone resorption, with enrichment of neutrophil extracellular traps (NETs) in periodontal tissues. DIC promoted myelopoiesis of hematopoietic stem and progenitor cells (HSPCs) by up-regulating the myeloid differentiation pathway. Intragastric administration of berberine dampened DIC and rescued the myeloid skewing of HSPCs, consequently alleviating periodontal destruction. Intriguingly, LIP induction after DIC remission still exhibited aggravated periodontal destruction and myeloid skewing of HSPCs, indicating a UC-trained immunity against periodontal damage. Conclusions Increased gut permeability and microbial dysbiosis in UC elevate the serum level of LPS and IL-1, inducing myeloid skewing of HSPCs with an immune memory. Generation of inflammatory potential myeloid cells causes NETs accumulation and aggravates periodontal destruction in the UC-related periodontitis. Lay Summary Ulcerative colitis exacerbates periodontitis by increasing gut permeability and microbial dysbiosis, elevating serum lipopolysaccharides and IL-1, and inducing immune memory in myeloid cells, leading to neutrophil extracellular traps accumulation and aggravated periodontal destruction. Graphical Abstract Graphical Abstract